•Cationic liposomes were produced at high lipid concentration.•Double hydrodynamic focusing produce smaller particle sizes when using high flow rates.•SAXS technique revealed the unilamellar nature ...of the cationic liposomes.
Microfluidics is a powerful technology that allows the production of cationic liposomes by the hydrodynamic focusing method. We first studied a single hydrodynamic focusing (SHF) device, which uses a central stream in which lipids dispersed in ethanol are injected and hydrodynamically compressed by the two aqueous streams. The ethanol diffusion from the inner stream to the aqueous stream encourages the formation of the liposomes. To intensify the mass diffusion and increase the surface area between the two fluids, a second device was designed with double hydrodynamic focusing (DHF). We investigated the influence of fluid flow velocity (Vf), Flow Rate Ratio (FRR) and total lipid concentration (Clip) on the particle size of the CLs produced. The DHF microfluidic device had the ability of using higher Vf values than the SHF device, which resulted in a higher productivity level. Small Angle X-ray Scattering (SAXS) experiments were performed to structurally characterize the cationic liposomes produced by both microfluidic devices. The SAXS results revealed that both devices produce unilamellar cationic liposomes with a very small fraction of multilamellar liposomes; this finding is in agreement with the observations made in the analysis of the liposomes using Transmission Electron Microscopy (TEM). The biological efficacies of the cationic liposomes produced by both microfluidic devices were examined in vitro in HeLa cells, which confirmed their potential for gene delivery and vaccine therapy applications.
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•High ionic strength enables the continuous synthesis of uniform stealth liposomes.•Monomodal size distribution is tuned by increasing ionic strength.•Diffusion coupled with ionic ...strength modulate lipid self-assembly kinetics.•Electrolytes shield the glass surface and prevent undesirable lipid aggregates.•Ionic strength can be exploited as a microfluidic process variable.
In this work, we describe a hydrodynamic flow-focusing microfluidic process to produce stealth cationic liposomes (SCL), stabilized with poly(ethylene glycol) (PEG), with uniform and reproducible features. Through cryogenic transmission electron microscopy (cryo-TEM) characterization and real-time monitoring, we verified the formation of multi-sized lipid self-aggregates, which can be attributed to micelles formation. These structures tend to undergo deposition within the PDMS/glass microchannels through intermolecular interactions with the glass walls, hindering not only the process reproducibility but also the final biological application of the SCL products. In view of this, we propose the modulation of ionic strength of the side streams aiming to ionically shield the glass surface, decrease the intermolecular interactions of the lipid polar heads, and, essentially, to promote the bilayer-driven self-assembly of SCL with 1% of DSPE-PEG2000 lipid. Herein, we applied phosphate-buffered saline (PBS) from 10 to 50 mM concentration as side streams, and evaluated its effects on SCL final physicochemical properties in terms of size distribution, mean diameter, zeta potential and polydispersity index (PDI). We present evidences indicating that the ionic strength can be used as a microfluidic process parameter to modulate the lipids self-assembly kinetics whilst preventing micelles formation. Finally, the proposed diffusion-based microfluidic system with high ionic strength enables the formation of monodisperse (PDI < 0.2) SCL of around 140 nm with monomodal size distributions and enhanced properties when compared to usual bulk mixing.
The Langhian Vc unit of Brielas section (Caparica road, Almada), in the marine Miocene of the Lower Tagus Basin (West Portugal), is rich in batoid species. In this study, batoids are represented by ...149 isolated fossil teeth and a single fossil dental plate collected from a bulk sample of washed and sorted sediment. A total of 12 species belonging to the orders Rhinopristiformes, Rajiformes, Torpediniformes and Myliobatiformes were identified. All genera and some species are known to have extant representatives. Aetobatus cappettai ANTUNES and BALBINO, 2006, is described now from Langhian material which suggests it is older than previously supposed. The biogeographic ranges, environmental constraints and behaviour of the species described in this study point out to an infralittoral environment characterized by the dominance of warm water conditions, where episodic upwelling contributed to the occurrence of different types of batoids coexisting in the same habitat. KEYWORDS Langhian. Chondrichthyes. Batomorphii. Brielas. Portugal.
Turbidite fragments collected by a fishing net off the central Portuguese coast (Peniche) present some fossils. The matrix is phosphatized and iron-rich with small quantities of manganese, zinc and ...copper. The occurrence of Megaselachus megalodon most probably excludes an age older than Middle Miocene. Its very advanced evolution stage is consistent with a Pliocene age. Based on planktonic foraminifera in depressions of cetacean skulls recovered in the same way, from the same area, the age of sharks and cetaceans is likely to range from latest Messinian to Early Pliocene. Condensed sedimentation is revealed by the co-occurrence of typical Late Pliocene and Quaternary foraminifera. Lack of benthic foraminifera suggests more or less deep environments, while a scallop, Mimachlamys varia, indicates nearby rocky substrate. The mako shark Isurus cf. oxyrhinchus is recorded in the area for the first time. The shark association represents a moderately warm environment as M. megalodon and Isurus are essentially temperate water dwellers, while no warm water form is known. Early Pliocene planktonic foraminifera point out to temperate to subtropical waters. Hence temperate to moderately warm conditions seem to have prevailed. KEYWORDS Megalodon. Mako shark. Planktonic foraminifera. Continental shelf. Age.
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•A microfluidic platform with two regions efficiently generated liposomes and lipoplexes.•The microfluidic device produced lipoplexes with significant reduced number of ...steps.•Microfluidic-obtained lipoplexes efficiently transfected mammalian cells.
In this work, pDNA/cationic liposome (CL) lipoplexes for gene delivery were prepared in one-step using multiple hydrodynamic flow-focusing regions. The microfluidic platform was designed with two distinct regions for the synthesis of liposomes and the subsequent assembly with pDNA, forming lipoplexes. The obtained lipoplexes exhibited appropriate physicochemical characteristics for gene therapy applications under varying conditions of flow rate-ratio (FRR), total volumetric flow rate (QT) and pDNA content (molar charge ratio, R±). The CLs were able to condense and retain the pDNA in the vesicular structures with sizes ranging from 140nm to 250nm. In vitro transfection assays showed that the lipoplexes prepared in one step by the two-stage configuration achieved similar efficiencies as lipoplexes prepared by conventional bulk processes, in which each step comprises a series of manual operations. The integrated microfluidic platform generates lipoplexes with liposome formation combined in-line with lipoplex assembly, significantly reducing the number of steps usually required to form gene carrier systems.
The mechanical environment of living cells is as critical as chemical signaling. Mechanical stimuli play a pivotal role in organogenesis and tissue homeostasis. Unbalances in mechanotransduction ...pathways often lead to diseases, such as cancer, cystic fibrosis, and neurodevelopmental disorders. Despite its inherent relevance, there is a lack of proper mechanoresponsive in vitro study systems. In this context, there is an urge to engineer innovative, robust, dynamic, and reliable organotypic technologies to better connect cellular processes to organ‐level function and multi‐tissue cross‐talk. Mechanically active organoid‐on‐chip has the potential to surpass this challenge. These systems converge microfabrication, microfluidics, biophysics, and tissue engineering fields to emulate key features of living organisms, hence, reducing costs, time, and animal testing. In this review, we intended to present cutting‐edge organ‐on‐chip platforms that integrate biomechanical stimuli as well as novel multicellular culture, such as organoids. We focused on its application in two main fields: precision medicine and drug development. Moreover, we also discussed the state of the art for the development of an engineered model to assess patient‐derived tumor organoid metastatic potential. Finally, we highlighted the current drawbacks and emerging opportunities to match the industry needs. We envision the use of mechanoresponsive organotypic‐on‐chip microdevices as an indispensable tool for precision medicine, drug development, disease modeling, tissue engineering, and developmental biology.
Microfluidics offers unique characteristics to control the mixing of liquids under laminar flow. Its use for the assembly of lipoplexes represents an attractive alternative for the translation of ...gene delivery studies into clinical trials on a sufficient throughput scale. Here, it was shown that the microfluidic assembly of pDNA/cationic liposome (CL) lipoplexes allows the formation of nanocarriers with enhanced transfection efficiencies compared with the conventional bulk-mixing (BM) process under high pDNA loading conditions. Lipoplexes generated by microfluidic devices exhibit smaller and more homogeneous structures at a molar charge ratio (R±) of 1.5, representing the ratio of lipid to pDNA content. Using an optimized model to fit small-angle X-ray scattering (SAXS) curves, it was observed that large amounts of pDNA induces the formation of aggregates with a higher number of stacked bilayers (N ∼ 5) when the BM process was used, whereas microfluidic lipoplexes presented smaller structures with a lower number of stacked bilayers (N ∼ 2.5). In vitro studies further confirmed that microfluidic lipoplexes achieved higher in vitro transfection efficiencies in prostate cancer cells at R ± 1.5, employing a reduced amount of cationic lipid. The correlation of mesoscopic characteristics with in vitro performance provides insights for the elucidation of the colloidal arrangement and biological behavior of pDNA/CL lipoplexes obtained by different processes, highlighting the feasibility of applying microfluidics to gene delivery.
Abstract To study whether treatment with 5′-adenosine triphosphate (ATP), an agonist of P2 purine receptors, attenuated intestinal dysfunction caused by ischemia (I) and/or reperfusion (R), rabbits ...were treated with ATP (15 mg · kg−1 , intravenously) or saline solution (SS) 60 minutes before I by occlusion of the superior mesenteric artery and/or R (120 minutes). After I or I/R isolated 2-cm jejunal segments were mounted in an organ bath to study nerve-mediated contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for optical microscopy. Compared to a sham group, the jejunal contractions were similar to sham hosts among I + ATP, but reduced in I + SS, I/R + SS, and I/R + ATP groups. The jejunal-enteric nerves were damaged in I + SS, I/R + SS, and I/R + ATP, but not the I + ATP group. These results suggested that ATP attenuated intestinal dysfunction produced by I, but not that caused by R.
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•Multifunctional pseudo-ternary nanocarriers are proposed.•Association of T-Rp3 protein with liposome increases the pDNA delivery efficiency.•Cell viability is negatively affected by ...association of pDNA/T-Rp3 complexes.
A major bottleneck in the development of efficient protocols for gene therapy and DNA vaccination is the low efficiency of gene transfer by non-viral vectors. This is mainly attributed to the fact that, during the traffic to target cell nuclei, vectors must overcome a series of enzymatic, physical, and diffusional barriers. The objective of this work was the development and characterization of new multifunctional non-viral vectors, based on proteins and lipids, that are able to efficiently deliver the foreign plasmid DNA (pDNA) to the nucleus of mammalian cells. A model pDNA containing the reporter gene GFP was complexed to protamine or the recombinant modular protein T-Rp3 to form binary complexes. In addition, we studied the ability of the cationic liposome composed of EPC/DOPE/DOTAP to encapsulate the binary complexes to form pseudo-ternary complexes (pDNA/protein/liposome). Characterization of the complexes were performed by dynamic light scattering (DLS), zeta potential, transmission electron microscopy (TEM) and pDNA accessibility assay. The assays revealed that both proteins were able to condense pDNA and form positively charged complexes, that could be efficiently encapsulated, leading to the formation of pDNA/protein/liposome complexes. Transfection studies using HeLa cells indicated that pDNA/protein formed by T-Rp3 were far more efficient for pDNA delivery than protamine. The complexes formed by pDNA/T-Rp3/liposome presented the highest transfection level (25%). On the other hand, cytotoxicity assays showed a significant decrease on cell viability when using pDNA/T-Rp3/liposome, indicating that the association of T-Rp3 with liposome significantly increase the delivery efficiency whilst prompting a proportional negative impact on cytotoxicity. A better understanding of the mechanisms of cell uptake and intracellular trafficking regarding the synergic effect between proteins and lipids in these vectors may, in the near future, lead to the development of more efficient non-viral vectors able to mimic the abilities of viral nucleic acid delivery.
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